High expression and production method of recombinant human thrombin in animal cell

A technology of human thrombin and thrombin, applied in the biological field, can solve the problems of rising production costs and low expression efficiency

Active Publication Date: 2012-09-26
SUZHOU ZELGEN BIOPHARML
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, when CHO cells are used for expression, the expression efficiency is low, leading to unfavorable factors such as increased production costs

Method used

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  • High expression and production method of recombinant human thrombin in animal cell
  • High expression and production method of recombinant human thrombin in animal cell
  • High expression and production method of recombinant human thrombin in animal cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1: Preparation of human prothrombin-2 gene

[0071] step one,

[0072] Using the extracted human hepatocyte mRNA as a template and Oligo-dT as a primer, the first-strand cDNA was synthesized by reverse transcription. Using cDNA as template, PCR reaction was carried out with high-fidelity DNA polymerase. Its primer sequence is as follows:

[0073] Primer 1: tgctgcggatccgcaaacgtcgtaccgccaccagtgagta (SEQ ID NO: 11);

[0074] Primer 2: atatgcggccgcctactctccaaactgatcaatga; (SEQ ID NO: 12)

[0075] Primer 3: tgctccagcgggtccggcgaaccgccaccagtgagtacca (SEQ ID NO: 13)

[0076] Primer 1 contains the C-terminal sequence of human protein C signal peptide and the N-terminal coding sequence of human prothrombin-2. Primer 2 contains the C-terminal coding sequence of human prothrombin-2 and introduces a NotⅠ restriction site. The chimeric gene of the peptide partial sequence and the complete coding region of human prothrombin-2, its sequence is as SEQ ID NO:1

[0077] P...

Embodiment 2

[0089] Example 2: Construction of Human Prothrombin-2 Expression Plasmid

[0090] The amplified product and the pGN vector (see Chinese patent 200910052576.1) were double-digested with KpnI and NotI respectively, and after the digested product was purified, the amplified product: pGN vector (molar ratio 3:1) was ligated with T4 DNA ligase to form ring. Construct the expression plasmids pGN / pc-PT2 and pGN / th-PT2, see the plasmid map image 3 .

Embodiment 3

[0091] Example 3: Using animal cells to express human prothrombin-2

[0092] The expression plasmid described in Example 2 was used to transfect conventional DHFR-deficient Chinese hamster ovary cells CHO DG44 (G Urlaub et al., somatic cells, Mol. Genet., 12, p555-556, 1986, hereinafter referred to as CHO).

[0093] The cells were cultured normally by adding hypoxanthine and thymine (HT) in commercial serum-free Excell-302 medium (Sigma Company), and the transfection was performed by electroporation. The process was as follows: CHO cells in logarithmic growth phase 24 hours before transfection, centrifuge to change the medium, resuspend with fresh medium and pipette into a single cell suspension; centrifuge the cells and follow the 10 7 Resuspend the cells at the density per milliliter, take 0.8 mL and put it into a 0.4 cm electric shock cup; add 200 micrograms of expression plasmid pGN / pc-PT2 or pGN / th-PT2, mix gently to avoid air bubbles; ice bath for 10 Minutes; electrot...

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Abstract

The invention provides a high expression and production method of recombinant human thrombin in animal cells. Specifically, the invention provides a fusion gene and a fusion protein coded by the same. The gene contains signal peptide sequence of human protein C and Gla structural domain deleted human prothrombin gene sequence, and can enhance expression level of human prothrombin-2. The invention also discloses a method for employing a recombinant expression vector containing the above nucleotide sequence to express and produce human prothrombin in animal cells. The invention also discloses a method for producing recombinant human thrombin by purification and activation. The recombinant human thrombin produced by the invention has biological activity satisfying treatment of correlative clinic diseases.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, it relates to a high-efficiency expression and production method of recombinant human thrombin in animal cells. Background technique [0002] Thrombin is a key enzyme in the body's blood coagulation system. It is produced in the damaged vascular endothelial cells and participates in various reaction steps of the blood coagulation process. Thrombin is a highly specific serine proteolytic enzyme that is activated by prothrombin. It consists of 308 amino acid residues and has a molecular mass of 36KD. Thrombin is a double-chain molecule containing an A chain and a B chain connected by a disulfide bond. The A-chain contains 49 amino acid residues, also known as the light chain; the B chain consists of 259 amino acid residues, also known as the heavy chain (Raimondo De Cristofaro et al. A natural prothrombin mutant reveals an unexpected influence of the A-chain's structure on the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/74C12N15/62C12N15/63C12N5/10
Inventor 张滨徐志刚盛泽林
Owner SUZHOU ZELGEN BIOPHARML
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